Interpretive Summary: Studies conducted in the Cereal Crops Research Unit are designed to improve barley grain for nutritional and malting. Important discoveries have been made in the past two years. Factors in the seed's endosperm (where starch is stored) were found to increase the production of an enzyme which breaks down starch. The gene which encodes the enzyme alpha-glucosidase (AGL) has been cloned from seeds. It is turned on when the seed germinates and helps to break down starch grains stored in the seed. AGL could have industrial utility in brewing and the production of food sweeteners, if it were more heat-stable. We will increase its heat-stability by changing the gene which encodes the chain of amino acids which make up the enzyme. The original gene was put into yeast, which then produced the enzyme. We have also cloned antifungal genes from seeds and are now putting them into barley to prevent infections by the Fusarium fungus, which devastates upper Midwest grain production. A new enzyme, belonging to a class of enzymes which break down stored seed protein during germination and malting, has been purified and characterized. In addition, a small protein which inhibits this group of enzymes has been purified. A method has been devised for analyzing several enzyme activities simultaneously, allowing the determination of each enzyme's contribution to the malting process. Also, several genes have been identified which may altered in a barley mutant which produces collapsed seeds. Identification of the altered gene may identify factors necessary for seed filling. These studies will lead to improvements in grain quality and help to ensure adequate supplies of high quality grain to producers and processors.

Technical Abstract:
Progress has been made over the past two years in four main areas. 1) Molecular studies were conducted to determine whether the starchy endosperm was found to increase production of high-pI alpha-amylase by prolonging the period of its synthesis and enhancing its secretion. Another study was conducted to clone and express genes which encode antifungal proteins (permatins) in the starchy endosperm of barley and oats. An oat clone was produced and used to transform Pichia pastoris yeast, which then produced permatin protein. We have also cloned and expressed (in Pichia) an alpha-glucosidase (AGL) gene. The transformed yeast produced the active enzyme. 2) A 31 kDa cysteine endoproteinase was purified from green malt. This enzyme readily hydrolyzes hordein, the main storage protein of barley seeds. An inhibitor (later found to be LTP1) was purified from green malt and found to inhibit all cysteine endoproteinases. 3) Studies of diastatic power determined that other enzymes, in addition to alpha-amylase, contribute strongly to diastatic power. A system has been devised for simultaneously analyzing the activities of four enzymes by path coefficient analysis. AGL produced in Pichia is being characterized as to its enzymatic properties. Efforts are underway to reengineer the gene to make a more heat-stable enzyme. 4) Differential gene screening techniques were used to find candidate genes responsible for the shrunken endosperm phenotype in the barley seg8 mutant. These are being tested by RNA blot and bulked segregant analysis.